Download Ketone bodies disturb fatty acid handling in isolated cardiomyocytes

753
Biochem. J. (2003) 371, 753–760 (Printed in Great Britain)
Ketone bodies disturb fatty acid handling in isolated cardiomyocytes
derived from control and diabetic rats
Danny M. HASSELBAINK, Jan F. C. GLATZ, Joost J. F. P. LUIKEN, Theo H. M. ROEMEN and Ger J. VAN DER VUSSE1
Department of Physiology, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands
According to the current paradigm, fatty acid (FA) utilization
is increased in the diabetic heart. Since plasma levels of
competing substrates such as ketone bodies are increased during
diabetes, the effect of those substrates on cardiac FA handling was explored. Cardiomyocytes were isolated from control
and streptozotocin-treated diabetic rats and incubated with
normal (80 µM) and elevated (160 µM) palmitate concentrations
in the absence or presence of ketone bodies, including acetoacetate (AcAc). Comparing control cardiomyocytes under normal
conditions (80 µM, no AcAc) with diabetic cardiomyocytes
(160 µM, 3 mM AcAc) showed that palmitate uptake was
−1
increased from 35.2 +
− 4.8 to 60.2 +
− 14.0 nmol · 3 min · g wet
weight−1 respectively. Under these conditions, palmitate oxidation
rates were comparable (58.9 +
− 23.6 versus 53.2 +
− 18.5 nmol ·
30 min−1 · g wet weight−1 ). However, in the absence of AcAc,
palmitate oxidation was significantly enhanced in diabetic cardiomyocytes, indicating that ketone bodies are able to suppress
cardiac FA oxidation in diabetes. The concomitantly increased
FA uptake in diabetic cells, mainly due to the elevated extracellular FA levels, may be responsible for the accumulation
of FA and triacylglycerol, as observed in the diabetic heart
in situ.
INTRODUCTION
EXPERIMENTAL
Diabetes mellitus is a clinical syndrome characterized by
hyperglycaemia caused by a relative or absolute deficiency of
insulin or by resistance to the action of insulin at the cellular level.
The diabetic condition leads to a significant elevation of blood
glucose and plasma lipids if left untreated [1,2]. Insulin deficiency
also results in increased hepatic synthesis of ketone bodies,
leading to elevated plasma levels of acetoacetate (AcAc) and
3-β-hydroxybutyrate (3HB) [3], which in turn can evoke serious
pathophysiological complications, such as diabetic ketoacidosis.
On the basis of studies on isolated hearts it was concluded that
cardiac long-chain fatty acid (FA) oxidation is increased in the
diabetic state [4–6]. These findings are, however, at variance with
observations on hearts in situ of human diabetic patients [7]. This
suggests that FA oxidation by the diabetic heart is governed by
factors present in the in situ situation, but absent when isolated
rat hearts are studied.
Under normal conditions, cardiac energy demand is mainly
covered by the oxidation of FA and glucose [8]. Lactate and
ketone bodies are also able to serve as oxidizable substrates [9].
It is tempting to state that the discrepancy between isolated hearts
and hearts of human diabetic patients with respect to cardiac FA
utilization is caused by the availability of alternative substrates,
such as ketone bodies.
In the present study we have tested the hypothesis that
ketone bodies, AcAc and 3HB, affect both uptake and intracellular handling of FA in diabetic cardiomyocytes. To this
end, cardiomyocytes were isolated from diabetic rats, 4 weeks
after streptozotocin (STZ) treatment. The cells were exposed
to AcAc, 3HB or a combination of both. FA uptake, oxidation,
and esterification were studied in the presence of normal
(physiological) or increased (diabetic) extracellular FA concentrations.
Treatment of animals
Key words: acetoacetate, β-hydroxybutyrate, oxidation, triacylglycerols, uptake.
Male Lewis rats were fed ad libitum, had free access to
water and were kept under a 12 h/12 h light/dark cycle [10].
The Institutional Animal Care and Use Committee, Maastricht
University, approved the experiments.
Animals (200–250 g body weight) were anaesthetized with
ketamine and xylazine (Eurovet, Bladel, the Netherlands), at
37.5 and 7.5 mg · kg−1 respectively via intraperitoneal injection.
Subsequently, the animals were treated with 70 mg · kg−1 STZ
in disodium citrate (100 mM, pH 4.5) via intravenous injection in
the tail vein to induce type 1 diabetes, and received a subcutaneous
injection of 2.5 ml of 20 % (w/v) glucose on both lateral sides.
Controls received vehicle only. STZ-treated animals were given
daily a 5 % glucose solution to drink. At 4 weeks after STZ
treatment, rats were anaesthetized with a subcutaneous injection
of 48 mg of sodium pentobarbital (CEVA Sante Animale B.V.,
Maassluis, the Netherlands) and 1000 i.u. of heparin. Thereafter,
hearts were surgically excised. In a subset of experiments
(n = 6) the anaesthetized animals did not receive heparin.
After thoracotomy, blood was collected from the left ventricle
with a citrate-rinsed syringe. Blood glucose was measured with
EuroflashTM (Lifescan, Milpitas, CA, U.S.A.) and plasma FA by
gas chromatography [11].
Isolation of cardiomyocytes
The cardiomyocyte isolation procedure was partially adapted
from that described by Luiken et al. [12]. The surgically removed
hearts were placed in ice-cold buffer A (containing, in mM:
115 NaCl, 2.6 KCl, 1.2 KH2 PO4 , 1.2 MgSO4 , 10 NaHCO3 , 10
Hepes, 11 glucose, pH 7.4). The aorta was mounted on a cannula
Abbreviations used: AcAc, acetoacetate; FA, fatty acid(s); FAT, fatty acid transporter; 3HB, 3-β-hydroxybutyrate; PL, phospholipids; STZ, streptozotocin;
TG, triacylglycerols.
1
To whom correspondence should be addressed (e-mail [email protected]).
c 2003 Biochemical Society
754
D. M. Hasselbaink and others
(diameter 2.0 mm) and hearts were perfused at 37 ◦C for 5 min
with buffer A at a flow rate of 7 ml · min−1 retrogradely as described by Langendorff [12a] to remove blood from the coronary
circulation. Thereafter, recirculating perfusion was started using
40 ml of buffer A supplemented with 0.7 % (w/v) BSA, 0.075 %
(w/v) collagenase and 15 mM butanedione monoxime. After
1 min, the flow rate was increased to 8 ml · min−1 and then
increased linearly to 14 ml · min−1 at 15 min after the start of the
recirculating perfusion. Flow was allowed to continue at this level
for 1 min. At time points 2 and 12 min 40 µl of 0.1 mM CaCl2 was
added to the perfusion buffer. At the end of the perfusion period,
the heart was removed from the cannula, transferred to a Petri
dish and carefully opened using forceps. The opened heart was
incubated with 10 ml of perfusate and 10 ml of buffer B (buffer
A supplemented with 0.2 mM Ca2+ and 2 % BSA) for 10 min
in a shaking waterbath (Aquatron ; HT Infors, Bottmingen,
Switzerland) (37 ◦C, 180 rev./min). The heart was subsequently
transferred to a Petri dish and was further dissected with forceps.
After dissection, the suspension was transferred to an Erlenmeyer
flask and placed in a shaking waterbath (37 ◦C, 180 rev./min)
for 5 min while the Ca2+ concentration was increased with
steps of 200 µM to 1 mM. Next, cell suspension was filtered
through a 0.2-mm-pore-size nylon mesh and cardiomyocytes
were pelleted by centrifugation (2 min, 17 g). The cell pellet was
resuspended in medium C (buffer A supplemented with 1 mM
Ca2+ and 2 % BSA) and centrifuged (2 min, 23 g), and this
procedure was repeated once. The cell pellet was resuspended
in 20 ml of medium C and transferred to a 50 ml conical tube.
Cardiomyocytes were allowed to rest for 1.5 h under mild rotation.
Prior to the start of the metabolic studies, cells were washed and
resuspended in medium C.
At the end of the metabolic studies, viability of the
cardiomyocytes was assessed by determining the percentage of
rod-shaped cells. Furthermore, cellular wet weight was obtained
by centrifugation in a microcentrifuge and subsequent removal of
the supernatant from samples taken at the end of the performed
experiments. All solutions used during the experiment were
continuously gassed (O2 /CO2 , 19 : 1).
performed as described by Luiken et al. [12]. Uptake experiments
were performed in triplicate for each condition tested.
Palmitate oxidation rate
The rate of palmitate oxidation was determined in the presence or
absence of ketone bodies during 30 min as described previously
[13]. The reaction was started by the addition of [1-14 C]palmitate
to the capped glass vial. After termination of the reaction,
production of 14 CO2 was measured after base trapping in NaOH.
The obtained 14 CO2 values were corrected for 14 CO2 trapped
in vials supplemented with [1-14 C]palmitate but in the absence
of cardiomyocytes in the incubation medium. Thereafter, this
value was normalized on mg wet weight of cardiomyocytes and
corrected for the percentage of non-rod-shaped cells. Oxidation
experiments were performed in triplicate for each condition
tested.
Incorporation of palmitate into intracellular lipids
A 2.0 ml suspension of cardiomyocytes was incubated in a capped
glass vial in the absence or presence of ketone bodies, in a
shaking waterbath (37 ◦C, 180 rev./min). The reaction was started
by the addition of [1-14 C]palmitate. After 30 min the reaction
was stopped as described by Luiken et al. [12]. Cells were
pelleted by centrifugation (2 min, 60 g, 4 ◦C) and washed twice
with 5.0 ml of ice-cold stop solution. The pellet was solubilized
in buffer containing (in mM), 50 Tris/HCl, pH 7.6, 150 NaCl,
5 EDTA, and 1 % (w/v) Triton X-100. Lipids were extracted with
chloroform/methanol and separated by TLC [11]. The lipid spots
corresponding to triacylglycerol (TG), phospholipids (PL) and
FA were scraped from the plate and the amount of radioactivity
was determined with a liquid-scintillation counter. Incorporation
experiments were performed in duplicate for each condition
tested.
Preparation of palmitate–BSA complex
Choice of substrate concentrations
To prepare a stock solution of palmitate–BSA complex, 100 µCi
of [1-14 C]palmitate and either 1.8 or 3.6 mM palmitate in 10 ml of
100 % ethanol were mixed with water containing KOH
(1.5 times the amount of palmitate on molar basis). After
evaporation of ethanol at 45 ◦C under nitrogen, the saponified
palmitate was added to 40 ml of buffer A supplemented with
2.5 % BSA. Prior to incubation, the stock solution was diluted
4.5-fold with buffer A supplemented with 1.0 mM CaCl2 .
Final extracellular concentrations of 80 µM and 160 µM
[1-14 C]palmitate were used. They correspond to the interstitial
concentrations of FA in healthy and diabetic rats respectively,
taking into account that the interstitial FA concentration is
approx. 60 % of the arterial FA level [14]. These FA conditions
were combined with various concentrations of AcAc, 3HB or a
combination of both ketone bodies. In general, in STZ-treated
rats, plasma concentration of ketone bodies is of the order of
3–6 mM [15,16], with a ratio of 3HB to AcAc of 2 : 1 [15].
Since AcAc was added to the incubation medium as the lithium
salt, the effect of 3 mM Li+ (as LiCl) was tested on FA uptake,
oxidation, and deposition of label in the various lipid pools. No
effect of Li+ on FA handling was found (results not shown).
Palmitate uptake rate
The rate of palmitate uptake was determined during 3 min
as described previously [13]. In short, a 2.0 ml suspension
of freshly isolated cardiomyocytes was incubated in a capped
glass vial (O2 /CO2 , 19 : 1) in the absence or presence of ketone
bodies, in an Aquatron shaking waterbath (37 ◦C, 180 rev./min).
After thermal equilibration, 0.5 ml of labelled palmitate (80 or
160 µM final concn.) was added and incubation allowed to
proceed for 3 min. Next, 2.0 ml of the incubation mixture was
transferred to a tube containing 8.0 ml of ice-cold stop solution
(buffer A supplemented with 0.1 % BSA, 1.0 mM CaCl2 and
200 µM phloretin). Correction for extracellular radioactivity was
c 2003 Biochemical Society
Materials
[1-14 C]Palmitic acid was obtained from NEN Life Science
Products (Boston, MA, U.S.A.). Collagenase type 2 (LS04176)
was from Worthington (Lakewood, NJ, U.S.A.), and phloretin,
BSA fraction V (A4503), STZ, LiCl, acetoacetic acid (lithium
salt), 3-β-hydroxybutyric acid, palmitic acid and butane-2,3dione monoxime was from Sigma (St. Louis, MO, U.S.A.).
Ketone bodies and cardiac fatty acid handling
755
Table 1 Initial palmitate uptake rate in control- and diabetic-rat
cardiomyocytes
Data are expressed as means +
− S.D. (n = 5). The asterisk (∗) indicates significantly different
from the value of the corresponding incubation with 80 µM palmitate (P < 0.05). Statistical
analysis revealed that the higher mean values in diabetic-rat cardiomyocytes as compared with
corresponding controls did not reach the level of significance. ‘6mM combination’ means a
mixture of 3HB (4 mM) and AcAc (2 mM). The final incubation medium contained (in mM)
NaCl (115), KCl (2.6), KH2 PO4 (1.2), MgSO4 (1.2), NaHCO3 (10), Hepes (10), glucose (11),
BSA (0.3), pH 7.4, palmitate and ketone-body concentrations were as indicated.
Initial uptake (nmol · 3 min−1 · g wet weight−1 )
[Palmitate] (µM)
Ketone bodies
Control
Diabetic
80
–
3 mM AcAc
3 mM 3HB
6 mM combination
35.2 +
− 4.8
33.4 +
− 3.9
36.0 +
− 8.0
36.0 +
− 5.0
40.1 +
− 7.6
39.3 +
− 9.0
41.2 +
− 9.4
41.4 +
− 12.8
160
–
3 mM AcAc
3 mM 3HB
6 mM combination
∗
54.8 +
− 5.0
∗
55.3 +
8.4
−
∗
60.7 +
7.6
−
64.0 +
10.1
−
∗
64.0 +
− 13.8
∗
60.2 +
14.0
−
∗
71.5 +
14.2
−
∗
70.1 +
27.5
−
Statistics
Results were obtained from at least four different cardiomyocyte
isolations and are presented as means +
− S.D. Analysis of
experiments within one group was performed with two-tailed
Student’s t tests. Comparison between groups was performed
with the one-way analysis of variance. In case the F ratio
obtained indicated that significant differences between groups
were present, a two-tailed Student’s t test for unpaired data
was carried out, applying Bonferroni’s adjustment for multiple
comparison [17]. For all analyses, the level of significance was
set at 0.05.
Figure 1
Influence of ketone bodies on the rate of FA oxidation
Cardiomyocytes derived from control and diabetic rats were incubated with [1-14 C]palmitate
for 30 min. For further details see the legend to Table 1. AcAc (3 mM) and 3HB (3 mM)
were either present separately or in combination [4 mM 3HB + 2 mM AcAc; ‘3HB/AcAc 6 mM
(2 : 1)’]. Data are expressed as means +
− S.D. (n = 6). Statistical significance : *P < 0.05,
versus corresponding absence of ketone bodies (−) ; #P < 0.05, control versus corresponding
diabetic : +P < 0.05, normal FA versus corresponding high FA.
RESULTS
General characteristics of STZ-induced diabetic rats
The body weight of the diabetic rats (301 +
− 49 g) was significantly
lower than that of controls (394 +
− 18 g). Diabetic plasma FA
levels were significantly higher than control (265 +
− 88 versus
59
µM).
Diabetic-rat
blood
glucose
concentrations
were
134 +
−
significantly higher than the control-rat values, namely 20.4 +
− 6.5
and 9.1 +
− 1.0 mM respectively.
Initial palmitate uptake rate
There was no difference in the initial uptake rate of palmitate
between control- and diabetic-rat cardiomyocytes at either
normal (80 µM) or elevated (160 µM) palmitate concentrations
(Table 1). However, increasing the palmitate concentration from
80 to 160 µM significantly enhanced its uptake. The percentage
of increase did not differ between control- and diabetic-rat
cardiomyocytes, being on the average 56 and 59 % respectively.
Ketone bodies did not affect the initial uptake rate of FA in
either control- or diabetic-rat cardiomyocytes, irrespective of the
palmitate concentration.
Palmitate oxidation rate
The rate of palmitate oxidation at 80 µM palmitate, in the
absence of ketone bodies, is about 56 % higher in diabetic-rat
cardiomyocytes than in those of control rats (P < 0.05) (Figure 1).
Increasing the palmitate concentration from 80 to 160 µM
resulted in a 70 % increase in oxidation rate in control-rat
cardiomyocytes (P < 0.05). The percentage increase in diabeticrat cardiomyocytes was comparable (68 %). Addition of 6 mM
ketone bodies (3HB/AcAc, 2 : 1) to control cardiomyocytes
significantly decreased the oxidation rate, both at normal and
elevated palmitate concentrations, by 51 and 58 % respectively.
Comparable inhibitory effects were seen in diabetic-rat
cardiomyocytes. Figure 1 also shows the differential effect of
3HB and AcAc. When cardiomyocytes were exposed to 3HB
(3 mM), the inhibitory effect was insignificant under all conditions
analysed. In contrast, exposure to 3 mM AcAc substantially
inhibited palmitate oxidation to a comparable extent in controland diabetic-rat cardiomocytes, at both palmitate concentrations.
Influence of various concentrations of ketone bodies
on FA oxidation
Figure 2 show that palmitate oxidation can be suppressed to
approx. 30 % of its maximal rate by AcAc. This is true for
cardiomyocytes derived from control and diabetic animals. The
maximal percentage of inhibition was found to be independent
from the two palmitate concentrations applied (Figure 2).
Half-maximum inhibition is reached at 0.3–0.5 mM AcAc and
c 2003 Biochemical Society
756
D. M. Hasselbaink and others
Figure 2
Influence of various concentrations of ketone bodies on the rate of FA oxidation
Control- and diabetic-rat cardiomyocytes were incubated with either 80 or 160 µM palmitate, both with various concentrations of ketone bodies, for 30 min. For further details see the legend to
Table 1. , AcAc; , 3HB/AcAc (2 : 1, mol/mol). Data are expressed as means +
− S.D. (n = 4).
•
maximum inhibition at 2 mM AcAc and higher. AcAc is therefore
a highly potent inhibitor of cardiomyocyte FA oxidation, and the
sensitivity to AcAc hardly differs between control- and diabeticrat cells.
The findings in Figure 2 also indicate that it is mainly AcAc
that causes the inhibitory effect of the mixture of AcAc and 3HB,
supporting the results shown in Figure 1.
Incorporation of labelled palmitate in various intracellular
lipid pools
In control cardiomyocytes, incubated under normal conditions
(80 µM palmitate, no ketone bodies), incorporation of labelled
palmitate in the cellular PL, TG and FA pool amounted to
−1
0.27 +
− 0.14, 1.46 +
− 0.80 and 0.07 +
− 0.05 nmol · 30 min · g wet
weight−1 respectively. In diabetic-rat cardiomyocytes, incubated
under diabetic conditions (160 µM palmitate, 3 mM AcAc)
substantially higher amounts of labelled palmitate were
incorporated in the PL, TG and FA pool, i.e. 0.69 +
5.15 +
− 0.32,
−
−1
−1
·
g
wet
weight
.
However,
3.02 and 0.19 +
0.18
nmol
·
30
min
−
c 2003 Biochemical Society
due to a relatively high degree of variation between the individual
experiments, the increase in the deposition of palmitate into the
various lipid pools in the diabetic cells did not reach the level
of significance. To evaluate the effect of varying the ambient
palmitate concentration and the addition of AcAc to the incubation
medium, the values obtained in either control or diabetic-rat cells
incubated in the presence of 80 µM palmitate, in the absence of
AcAc, was set at 1.0. This procedure enabled us to use the cells as
their own control. Raising the concentration from 80 to 160 µM
tended to increase the incorporation of palmitate in the PL and
TG pool of control cells (Figure 3). The increased deposition of
label in the PL, TG and FA pool of diabetic cardiomyocytes was
highly significant when the extracellular palmitate concentration
was increased from 80 to 160 µM (P < 0.05).
Exposure of cardiomyocytes, either derived from control or
diabetic hearts, to 3 mM AcAc at 80 µM palmitate did not affect
the incorporation of palmitate in the PL and TG pool. The FA pool
in diabetic cardiomyocytes incubated in the presence of 80 µM
palmitate showed a significant expansion when exposed to 3 mM
AcAc. This effect was not seen in control-rat cardiac muscle
cells. When the concentration of palmitate was increased from
Ketone bodies and cardiac fatty acid handling
757
DISCUSSION
The present observations indicate that ketone bodies, in particular
AcAc, affect FA handling in the cardiac muscle cell. This finding
is of interest because it allows one to fully appreciate the changes
in cardiac FA utilization in the diabetic heart, since the circulating
concentration of ketone bodies is substantially increased in this
pathophysiological condition. AcAc causes a mismatch between
FA uptake (not affected by AcAc, but increased due to enhanced
supply) and FA oxidation (significantly inhibited by AcAc)
when diabetic cardiomyocytes, derived from STZ-treated rats and
incubated under diabetic conditions, i.e. 160 µM palmitate, 3 mM
AcAc, were compared with control cardiomyocytes, incubated
under normal conditions, i.e. 80 µM palmitate, no ketone bodies.
This mismatch most likely explains the accumulation of lipids,
FA and TG in the diabetic heart in situ. Moreover, the present
findings are challenging the current paradigm that FA oxidation
is enhanced in the diabetic-rat heart, since this notion is based
on experiments performed on isolated hearts perfused with media
devoid of ketone bodies.
General background
Figure 3 Deposition of radiolabelled palmitate in various lipid pools in
control- and diabetic-rat cardiomyocytes
After incubation for 30 min with radiolabelled palmitate, lipids were extracted and subjected
to TLC. Data are presented relative to the values obtained in control- and diabetic-rat
cardiomyocytes respectively, incubated in the presence of 80 µM palmitate without ketone
bodies. For further details see the legend to Table 1. The asterisk () indicates significantly
different from the corresponding incubation with 80 µM palmitate, without ketone bodies. Data
are expressed as means +
− S.D. (n = 4).
80 to 160 µM in the incubation medium of control- or diabeticrat cells, exposed to 3 mM AcAc, the deposition of labelled
palmitate tended to increase (control), or significantly increased
(diabetic-rat cells), in the PL and TG pool. Since this pattern
was also observed in cells incubated in the absence of AcAc,
increased deposition of labelled FA in the esterified lipid pools
is mainly caused by increased availability of FA rather than by
a modulating effect of AcAc. The same conclusion holds for the
FA pool in control cardiomyocytes. In contrast, in diabetic-rat
cardiomyocytes, bulk accumulation of labelled palmitate in the
FA pool seems to be mainly caused by the presence of AcAc in
the incubation medium.
Earlier findings in hearts isolated from STZ-treated rats and
immediately freeze-clamped for tissue lipid analysis indicated a
4.5-fold accumulation of FA in the diabetic-rat heart, while blood
FA levels were enhanced only two-fold [11]. Accumulation of FA
in cardiac tissue, also reported by Heyliger and colleagues [18],
strongly suggests a mismatch between the uptake of extracellular
FA and their intracellular utilization. A common feature in patients
suffering from diabetes and in experimental diabetic animals is an
increased circulatory level of ketone bodies, AcAc and 3HB [19].
This increase is most striking in diabetic ketoacidosis, a major
complication of type 1 diabetes. Elevated levels of ketone bodies
have been also observed in poorly controlled type 2 diabetes,
chronic cardiac failure and during the consumption of high-fat
diet [20–23]. Pioneering studies of Randle and colleagues have
shown that ketone bodies serve as alternative substrates for energy
conversion in cardiac muscle [19]. Considering the enhanced
availability of ketone bodies in the diabetic state, it is likely that
the presence of those substances interferes with myocardial lipid
metabolism.
To fulfil cardiac energy requirements, two main nutrients, FA
and glucose, are continuously supplied to the heart, extracted from
the extracellular compartment and oxidized in the mitochondrial
matrix to regenerate the myocytal ATP pool. Under normal
circumstances, oxidation of plasma-borne FA contributes up to
60–70 % of energy conversion in the healthy heart [8]. In the
diabetic-rat heart, fuel selection is significantly changed, but
the direction and magnitude of the alterations in cardiac FA
consumption appear to differ greatly between studies dealing
with this subject matter [8]. These considerations prompted us to
investigate the effect of ketone bodies on FA handling by cardiac
muscle cells derived from STZ-treated rats.
Initial palmitate uptake rate
The present findings clearly indicate that the rate of palmitate
uptake by isolated cardiomyocytes is not changed in the
diabetic state when the cells were exposed to normal extracellular palmitate concentrations, i.e. 80 µM. Elevation of the
concentration to 160 µM (diabetic conditions) caused a significant
and comparable increase in the initial rate of palmitate uptake,
both in diabetic- and control-rat cardiomyocytes, indicating that,
under the present conditions, trans-sarcolemmal FA transport
c 2003 Biochemical Society
758
D. M. Hasselbaink and others
Figure 4 Initial uptake and oxidation of palmitate by control- and diabeticrat cardiomyocytes
This Figure is a compilation of data shown in Table 1 and Figure 1. Control cardiomyocytes
were incubated for either 3 min (initial uptake) or 30 min (oxidation) under normal conditions,
i.e. 80 µM palmitate, in the absence of AcAc; diabetic-rat cardiomyocytes were incubated for
similar time intervals under diabetic conditions, i.e. 160 µM palmitate in the presence of 3 mM
AcAc. Data are expressed as means +
− S.D. (n = 5 or 6). The asterisk () indicates a statistically
significant difference between control and diabetic cells.
is not altered in diabetic-rat cells. This result might appear
surprising, since previous studies had shown that STZ treatment
resulted in an increase in the myocardial content of proteins
known to facilitate FA uptake, including fatty acid transporter
(FAT)/CD36 [24,25]. A possible explanation is that the increased
amount of FAT/CD36 is stored in the cytoplasmic compartment
rather than incorporated into the plasma membrane.
The fact that neither AcAc nor 3HB, present in the incubation
medium, altered the uptake rate of palmitate in cardiomyocytes
indicates that the mechanisms underlying the unidirectional influx
of FA across the sarcolemma are insensitive to the presence of
ketone bodies in the interstitial compartment. Since diabetic-rat
cardiomyocytes in vivo are exposed to both elevated FA levels and
enhanced concentrations of ketone bodies, the conclusion can be
drawn that diabetic-rat cardiomyocytes take up higher amounts of
FA from their surroundings than do the control, merely because
the extracellular FA availability is increased (Figure 4).
weight−1 ; 80 µM palmitate, no ketone bodies) (Figure 4). It is
noteworthy that the inhibitory effect of ketone bodies on myocyte
FA oxidation is almost exclusively caused by AcAc, since the
effect of the reduced counterpart, 3HB, was found to be negligible.
The lack of effect of 3HB is remarkable, considering that both
3HB and AcAc can be used as oxidizable substrate by the heart
[19]. At present no conclusive explanation can be offered for
this intriguing observation, although differences in redox state in
the cardiomyocytes exposed to either AcAc or 3HB should be
considered. In this respect, the previously obtained findings by
Isales and colleagues are worth mentioning, as they also observed
differential effects of 3HB and AcAc, namely on the regulation
of growth factors in brain endothelial cells [26].
Detailed analysis of the inhibitory effect of AcAc on
cardiomyocyte palmitate oxidation revealed that, both in controland diabetic-rat cells, AcAc is a potent inhibitor of palmitate
oxidation. The maximal degree of inhibition in both cell types was
on the order of 70 %, irrespective of the concentration of palmitate
in the extracellular fluid. Since ketone bodies are permeant to cells
via the H+ /monocarboxylate co-transporter [27–29], transport
limitations of ketone bodies through this protein-mediated process
could therefore contribute to the inability to depress FA oxidation
any further.
Half-maximal inhibition was obtained at about 0.3–0.5 mM
AcAc, indicating that, on the one hand, FA oxidation by
cardiomyocytes is highly sensitive to AcAc in the surrounding
medium, since this relatively low concentration of AcAc is readily
reached under a variety of conditions [30] and, on the other,
that the sensitivity to AcAc did not differ between diabeticrat and healthy-rat cardiomyocytes. The latter indicates that
cardiomyocytes in diabetic-rat hearts do not adjust to chronically
elevated levels of ketone bodies by altering their sensitivity
towards the inhibitory action of these compounds. This kind of
adjustment might have been expected, since previous observations
by Grinblat and colleagues [31] and Kante and co-workers [32]
showed that the expression and activity of two enzymes
involved in ketone-body handling, namely 3-β-hydroxybutyrate
dehydrogenase and 3-oxoacid-CoA transferase, are significantly
decreased in the diabetic-rat heart as compared with controls.
Obviously, not merely the intracellular conversion, but also other,
as yet unidentified properties may be involved in the inhibitory
action of ketone bodies on cardiac FA oxidation.
Palmitate oxidation rate
The rate of palmitate oxidation was found to be significantly
higher in cardiomyocytes isolated from diabetic-rat hearts than
in control cardiomyocytes when incubated under comparable
normal conditions (80 µM palmitate, no ketone bodies). These
findings support some earlier observations in isolated rat hearts,
namely that the rate of FA oxidation is increased in hearts
obtained from diabetic animals [4,6]. Increasing the extracellular
palmitate concentration from 80 to 160 µM enhanced the
FA oxidation rate about 70 % both in control and diabetic
cardiomyocytes. Comparing control cells at normal ambient FA
levels with diabetic-rat cardiomyocytes subjected to diabetic
FA concentrations revealed that the rate of palmitate oxidation
is approx. 3 times higher in the latter (Figure 1). However, this
picture substantially changes when ketone bodies were added
to the incubation medium to mimic the diabetic conditions
as much as possible. Co-incubation of diabetic-rat cardiac
muscle cells with 160 µM palmitate and 3 mM AcAc inhibited
palmitate oxidation by approx. 65 %. The resulting oxidation rate
−1
−1
(53.2 +
− 18.5 nmol · 30 min · g wet weight ) was not significantly different from that measured in control cells incubated
−1
under control conditions (58.9 +
− 23.6 nmol · 30 min · g wet
c 2003 Biochemical Society
Incorporation of palmitate in cellular lipid pools
In general, the amount of labelled palmitate sequestered in the
esterified lipid pool (TG and PL) depended on the extracellular
FA concentration. Increasing the supply of palmitate resulted in
a higher degree of esterification, both in control- and diabetic-rat
cardiomyocytes. AcAc exerted no significant effect on the rate of
palmitate esterification under these conditions. The observation
that the TG content is enhanced in the diabetic-rat heart in situ
[33] is supported by the present findings. It is noteworthy that
the increased supply of extracellular FA, rather than the presence
of ketone bodies, is instrumental in the enhanced deposition of
palmitate into the intracellular TG pool (Figure 3). Previous
studies also indicated that ketone bodies are able to inhibit
lipolysis [34], which may contribute to the net expansion of the
TG pool in the diabetic-rat heart in situ. The present findings,
however, do not support this notion, since (additive) effects of
AcAc on the incorporation of palmitate into the intracellular TG
pool were absent.
The present study clearly shows that, in diabetic-rat
cardiomyocytes AcAc increases the intracellular level of nonesterified palmitate, both at the extracellular concentration of
Ketone bodies and cardiac fatty acid handling
80 and 160 µM palmitate (Figure 3). It is noteworthy that the
4-fold increase in deposition of labelled palmitate in the cellular
FA pool matches very well with the 4.5-fold increase in the FA
content of diabetic hearts in situ [11]. Collectively, the present
findings strongly suggest that accumulation of FA in cardiac
tissue is caused by a combination of two separate changes in the
diabetic cardiomyocyte: the initial uptake rate of FA by diabeticrat cardiomyocytes is enhanced, due to the increased supply of
extracellular palmitate, on the one hand, and the inhibitory action
of ketone bodies, in particular AcAc, on the intracellular rate of
FA oxidation, on the other. This notion is illustrated in Figure 4,
which summarizes the results shown in Table 1 and Figure 1. It
should be emphasized, however, that the present data are obtained
in resting cardiac muscle cells. It would be interesting to explore
how, at enhanced workload (e.g. the beating heart), circulating
ketone bodies interact with cardiac FA handling.
Possible implications of the present findings
One may speculate on the pathophysiological consequences of
chronically elevated FA levels inside the cardiomyocytes of the
diabetic-rat heart. Pioneering studies of Feuvray and colleagues
have shown that accumulation of lipotoxic FA and their intermediates are associated with mechanical dysfunction and cell
damage in diabetic hearts subjected to ischaemia [35]. Furthermore, other studies indicated that FA at elevated concentrations
exert cytotoxic effects, including aberrations in signaltransduction cascades [36–38]. More recent observations by Van
der Lee and colleagues [10] and others [39–41] have convincingly
shown that chronic exposure of cardiomyocytes to FA modulates
the expression of a panel of cardiac enzymes involved in
carbohydrate and fatty acid handling. The down-regulation of
hexokinase II and the up-regulation of long-chain acyl-CoA
dehydrogenase, especially, may explain the decline in glucose
utilization and enhanced FA oxidation commonly observed in
isolated hearts obtained from STZ-treated diabetic rats [10].
The present findings, however, challenge the current paradigm
that FA oxidation is enhanced in the intact diabetic heart [42,43].
The latter notion is based on experimental findings showing a
significant increase in the oxidation rate of radiolabelled palmitate
by hearts isolated from STZ-treated rats [44]. Results of other
studies, however, do not support these findings. The rate of FA
oxidation was found to be decreased in hearts from spontaneously
diabetic rats [43], and unchanged in hearts obtained from insulinresistant obese rats [45]. Moreover, the utilization of FA by the
hearts in vivo of diabetic swine was found to be not significantly
different from control [44], while the uptake and oxidation of
iodine-labelled FA analogues by hearts of patients with impaired
glucose tolerance was decreased [7]. Finally, cardiac FA uptake
and oxidative utilization of patients suffering from type 1 and
type 2 diabetes did not significantly differ from healthy controls
[7].
Since the contrasting findings are most pronounced between
isolated hearts (enhanced FA utilization) and intact hearts in situ
(no changes or even a decline in FA uptake and/or oxidation), the
regulatory role of other factors present in blood plasma, but not in
buffers used in perfusion experiments, on isolated hearts should be
considered. These factors may be, among others, elevated blood
concentration of ketone bodies in the intact experimental animal
or subjects suffering from diabetes.
The present findings might also have ramifications for other
conditions associated with enhanced plasma ketone-body levels,
such as starvation, high-fat diets and cardiac failure. Under these
conditions cardiac FA handling may be compromised by elevated
exposure of the heart to circulating ketone bodies.
759
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